The identification of cell cycle regulatory proteins has been greatly facilitated by studies of mutant yeast strains with abnormalities related to cell proliferation. Among the gene products defined in yeast is Far 1 (1), whose mammalian homologue, p21, alters the activity of cyclin-dependent kinases and is implicated in cell cycle progression and senescence (2-13). p21, also known as WAF1, CIP1 or SDI1 (11,12,14,15), is a downstream target of the p53 tumor suppressor gene and has thus been implicated indirectly in malignant transformation (15-18). Induction of p53 in response to DNA damage results in G1 checkpoint arrest (16-19), at which point DNA repair is accomplished prior to DNA replication in S phase. Consistent with its presumed role as a downstream effector for p53 , p21 has been shown to inhibit proliferating cell nuclear antigen (PCNA) dependent DNA replication but not DNA repair in vitro (20).
Zhang et al, Genes & Development (1994) 8:1750) studied p21 in vitro. As p21 functions as a kinase inhibitor, it had been predicted that normal cells should contain virtually no active cyclin kinases. By demonstrating that p21-containing cyclin kinases exist in both active and inactive states, Zhang et al rationalized that p21 was involved in controlling cell cycle progression in normal cells. Zhang et al found that in fibroblasts transformed with a variety of tumor viral oncoproteins, cyclin kinases exist in a binary state [cylcin/CDK]; whereas in normal fibroblasts multiple cyclin kinases exist in quaternary complexes containing p21 [cyclin/CDK/ proliferating cell nuclear antigen (PCNA)/p21]. Active complexes contain a single p21 molecule. In contrast inactive complexes possess multiple p21 subunits. Although changes in p21 stoichiometry were sufficient to account for the conversion of active to inactive complexes in vitro, Zhang et al believed that "association of cyclin knases with p21 must be intertwined with other modes of regulation in vivo." Zhang et al noted that "it is not known what effect association with noninhibitory levels of p21 might have on the function of these CDK-modifying enzymes in vivo."
WO 94/09135 describes methods and diagnostic kits for diagnosing transformation of a cell, involving detection of the subunit components of cyclin complexes. In particular, the method pertains to the interaction of cyclins, PCNA, CDKs and low molecular weight polypeptides such as p21, p19 and p16.
Despite the evidence of cyclin kinase inhibitory activity in vitro, the role of p21 in tumor formation and its ability to reverse the malignant phenotype in vivo has not been defined.